Mitochondria play a key role in linking cell respiration to cell survival and are critical elements in many age- related degenerative pathologies: The mitochondrial theory of aging proposes that oxidative damage leads to irreversible mitochondrial dysfunction and tissue degeneration with age. New non-invasive methods developed in the last grant cycle have revealed significant mitochondrial uncoupling measured in vivo in aged mouse and human skeletal muscle that is at least partially reversible. This proposal builds on these findings to evaluate: 1) the mechanisms underlying in vivo mitochondrial dysfunction with age, and 2) the reversibility of each component of mitochondrial dysfunction. We employ state-of-the-art optical and magnetic resonance spectroscopic approaches to quantify in vivo deficits in mitochondrial ATP and Oz fluxes with age. The biochemical bases of these deficits are determined from in vitro tissue analysis of the same mouse muscles. We study wild-type mice over a range of ages to evaluate how accumulation of oxidative damage is related to mitochondrial defects and dysfunction in natural aging. Transgenic models with altered antioxidant activities and uncoupling protein (UCP3) expression are used to identify the underlying mechanisms of this dysfunction. Aim 1 tests the roles of oxidative damage and uncoupling protein activity in the loss of mitochondrial coupling (reduced P/O) with age in wild-type and transgenic mice. Aim 2 determines how respiratory chain defects impair respiratory function in vivo. We pair in vivo measurements of 62 flux with measures of oxidative damage to specific mitochondrial components at multiple ages in wild- type and transgenic mice. Aim 3 tests the reversibility of the mitochondrial defects and dysfunction measured in Aims 1 and 2. Endurance exercise is used to increase mitochondrial proliferation and turnover, thereby replacing damaged mitochondria and improving function. The relevance of the proposed research to human health is two-fold. 1) The determination of the specific biochemical mechanisms leading to mitochondrial dysfunction will identify potential strategies to retard or reverse mitochondrial pathologies with age. 2) Our long-term goal is the development of non- invasive methods to diagnose mitochondrial dysfunction and follow the progress of interventions meant to reverse disability in the elderly.